A typhus group-specific protease defies reductive evolution in rickettsiae

Abstract

Phylogenomics reveals extreme gene loss in typhus group (TG) rickettsiae relative to the levels for other rickettsial lineages. We report here a curious protease-encoding gene (ppcE) that is conserved only in TG rickettsiae. As a possible determinant of host pathogenicity, ppcE warrants consideration in the development of therapeutics against epidemic and murine typhus.

FIG. 1.

Prolyl oligopeptidase (POP) genes of rickettsiae. (A) TG-specific genes identified previously (16) or in this study. Annotation is from PATRIC (32). (B) Tertiary structure of Myxococcus xanthus prolyl endopeptidase (31), illustrating the major features of POP proteins: blue, β-propeller domain; yellow, α/β hydrolase fold domain; red, catalytic triad (Ser-Asp-His); cyan, substrate. (C) Distribution of POP genes across 15 rickettsial genomes. Phylogeny was estimated from 18 genes encoding the rvh type IV secretion system as previously described (14). Branch support is from 1 million bootstrap replications; asterisks indicate 100% bootstrap support. The rickettsial classification scheme (15, 16) was as follows: red, ancestral group; turquoise, TG; blue, transitional group; and brown, SFG. Ticks are listed as the principal arthropod vector for all species except R. prowazekii (louse), R. typhi (flea), R. felis (flea), and R. akari (mite). Genome statistics were compiled from the PATRIC database (32). ptrB, oligopeptidase B gene (EC 3.4.21.83); ppcE, postproline cleaving enzyme gene (EC 3.4.21.26); dap2, dipeptidyl aminopeptidase 2 gene (EC 3.4.19.1). Full-length POP genes are colored light green, with truncated and split open reading frames colored black. Skulls and crossbones symbols depict gene fragments (three or more) detected with a TBLASTN search against the NCBI Rickettsiales database (taxid:766). Rickettsial palindromic element 3 (RPE-3) is depicted with a yellow ball-and-stick at its approximated insertion point in all derived SFG rickettsiae.

FIG. 2.

Predicted sequence locations of β-propeller blades within R. typhi PpcE and PtrB. Using as queries those sequence regions corresponding to the β-propeller domains of bacterial prolyl endopeptidases of known structure, PSI-BLAST (1) iterative searches of the NCBI NR database were preformed with a very conservative E value cutoff of 10⁻⁴⁰. Those database sequence regions homologous to the queries were pooled, and both closely related sequences (those sharing ≥80% identity) and sequences less than 315 amino acids in length were removed. The remaining 380 sequence regions were used as input to a Bayesian Markov chain Monte Carlo sampling procedure for detecting subtly conserved internal repeats (22-24). This resulted in the detection and alignment of 2,891 putative β-propeller blades, including those shown in the alignment in panel A, which corresponds to regions within the R. typhi PpcE and PtrB sequences and to β-propeller blades of known structure within prolyl endopeptidases from Myxococcus xanthus (31) (as indicated in panel B), Sus scrofa (11), and Novosphingobium capsulatum (31) (Protein Data Bank accession numbers 2BKL, 1QFS, and 1YR2, respectively). The seven propeller blades are labeled in the N- to C-terminal direction as B1 to B7 in panel A and are colored purple, magenta, scarlet, red, orange, yellow, and green, respectively, in panel B. Note that the Bayesian sampler failed to detect the putative blade B4 within R. typhi PpcE (as indicated by an asterisk); instead, an optimal alignment of this region against a profile of the detected repeats was obtained independent of the sampler. The most commonly occurring residues at each position (within all 2,891 blades) are shown directly below the alignment, and directly below these, the corresponding (weighted) residue frequencies are given in integer tenths; for example, “2” indicates that the corresponding pattern residue occurs in 20% to 30% of the aligned sequences at that position. The most commonly occurring residue types are highlighted in the alignment, with chemically similar residues colored similarly, as previously described (21). The β-propeller domains in panel B are oriented perpendicularly to the structure shown in Fig. 1B. (C) Gene expression of R. typhi PpcE and PtrB in HeLa cells was as previously described (2). On the basis of Student's t test, the P value (for RT0165 versus RT0272) was 0.022503 (P < 0.05). The ability of the proximal N-terminal sequence of PpcE to translocate E. coli alkaline phosphatase (PhoA) to the periplasm is illustrated.